Pulse caller and base caller

ABSTRACT

System and methods for identifying nucleotides based on data acquired from a sensor during sequencing of nucleic acids. The method may include obtaining characteristics of light detected from luminescent labels associated with the nucleotides during nucleotide incorporation events. The characteristics may include, for each nucleotide incorporation event, a temporal characteristic the light and an intensity characteristic of the light. The temporal characteristic representing a speed of decay of a probability of photon emission by a luminescent label after excitation. The method may further include grouping points representing the characteristics of the nucleotide incorporation events into groups of points. The individual points may represent at least the temporal characteristic and the intensity characteristic for a corresponding nucleotide incorporation event. The method may further include assigning the groups of points to individual nucleotides.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application62/343,997, titled “PULSE CALLER AND BASE CALLER,” filed Jun. 1, 2016,which is hereby incorporated by reference in its entirety.

BACKGROUND

Sequencing of nucleic acids (e.g., deoxyribonucleic acid (DNA),ribonucleic acid (RNA)) includes identifying individual of nucleotidesin a target nucleic acid. Some nucleic acid sequencing methods includeidentifying individual nucleotides as they are incorporated into nucleicacid strand complementary to the target nucleic acid. The series ofnucleotides for the complementary strand identified during thesequencing process may then allow for identification of the nucleotidesequence for the target nucleic acid strand.

SUMMARY

Some embodiments relate to a method that includes receiving firsttime-bin information regarding the times at which a first luminescentlabel emits light in response to excitations of the first luminescentlabel, calculating first light intensity information based on the firsttime-bin information, receiving second time-bin information regardingthe times at which a second luminescent label emits light in response toexcitations of the second luminescent label, calculating second lightintensity information based on the second time-bin information, andcalculating times at which nucleotide incorporation events occur usingthe first and second light intensity information.

The calculating of the times at which the nucleotide incorporationevents occur may be performed using a pulse identification algorithm.The pulse identification algorithm may include a changepoint algorithm,a running mean/median and variance algorithm or a state machinealgorithm. Calculating the first light intensity information may includesumming the first time-bin information, and calculating the second lightintensity information may include summing the second time-bininformation.

Some embodiments relate to a method that includes receiving firsttime-bin information regarding times at which a first luminescent labelemits first light in response to excitations of the first luminescentlabel and calculating a first temporal characteristic of the first lightbased on the first time-bin information. The temporal characteristic mayrepresent a speed of decay of a probability of photon emission by thefirst luminescent label after excitation. The method may further includereceiving second time-bin information regarding times at which a secondluminescent label emits second light in response to excitations of thesecond luminescent label and calculating a second temporalcharacteristic of the second light based on the second time-bininformation. The second temporal characteristic may represent a speed ofdecay of a probability of photon emission by the second luminescentlabel after excitation. The method may further include calculating timesat which nucleotide incorporation events occur using the first andsecond temporal characteristics.

The calculating of the times at which the nucleotide incorporationevents occur may be performed using a pulse identification algorithm.The pulse identification algorithm may include a changepoint algorithm,a running mean/median and variance algorithm or a state machinealgorithm.

Some embodiments relate to a method that includes determining one ormore temporal characteristics representing a speed of decay of aprobability of photon emission by one or more luminescent labels afterexcitation and calculating times at which nucleotide incorporationevents occur using the at least one temporal characteristic.

The calculating of the times at which nucleotide incorporation eventsoccur may be also performed using an intensity of light emitted by theone or more luminescent labels.

Some embodiments relate to a method that includes receiving time-bininformation regarding times at which a luminescent label emits light inresponse to excitations of the luminescent label, calculating lightintensity information based on the time-bin information, and calculatinga time at which at least one nucleotide incorporation event occurs usingthe light intensity information.

The time at which at least one nucleotide incorporation event occurs maybe also performed using a temporal characteristic of the light.

Some embodiments relate to a method of identifying nucleotides thatincludes obtaining characteristics of light detected from luminescentlabels associated with the nucleotides during nucleotide incorporationevents. The characteristics may include, for each nucleotideincorporation event: i) a temporal characteristic the light, thetemporal characteristic representing a speed of decay of a probabilityof photon emission by a luminescent label after excitation; and ii) anintensity characteristic of the light. The method may further includegrouping points representing the characteristics of the nucleotideincorporation events into groups of points, individual pointsrepresenting at least the temporal characteristic and the intensitycharacteristic for a corresponding nucleotide incorporation event, andassigning the groups of points to individual nucleotides.

The temporal characteristic may include a luminance lifetime or a ratioof photons detected in different time bins. The grouping of the pointsmay be performed by a clustering algorithm. The clustering algorithm mayperform k-means clustering in which k is greater than or equal to four.The individual groups of points may be assigned to individualnucleotides based on predetermined light-emitting characteristics of theluminescent labels.

Some embodiments relate to a method of calibrating a sequencinginstrument, the method includes obtaining characteristics of lightdetected from luminescent labels associated with the nucleotides duringnucleotide incorporation events. The characteristics may include, foreach nucleotide incorporation event: i) a temporal characteristic thelight, the temporal characteristic representing a speed of decay of aprobability of photon emission by a luminescent label after excitation;and ii) an intensity characteristic of the light. The method may furtherinclude grouping points representing the characteristics of thenucleotide incorporation events into groups of points, individual pointsrepresenting at least the temporal characteristic and the intensitycharacteristic for a corresponding nucleotide incorporation event,assigning individual groups of points individual nucleotides,determining one or more criteria distinguishing the groups of points,and storing the one or more criteria.

The one or more criteria may include one or more boundaries between thegroups of points. The one or more criteria may include centroids of thegroups of points. The one or more criteria may be stored in non-volatilememory. Grouping the points may include running a clustering algorithmon the points.

Some embodiments relate to a method of identifying nucleotides, themethod includes obtaining characteristics of light detected fromluminescent labels associated with the nucleotides during nucleotideincorporation events. The characteristics may include, for eachnucleotide incorporation event: i) a temporal characteristic of thelight, the temporal characteristic representing a speed of decay of aprobability of photon emission by a luminescent label after excitation;and ii) an intensity characteristic of the light. The method may furtherinclude assigning the nucleotide incorporation events to nucleotides byevaluating the temporal characteristic and the intensity characteristicin view of stored criteria for a sequencing instrument distinguishingbetween the characteristics of the light for the luminescent labels.

The stored criteria may include one or more boundaries betweencharacteristics of the luminescent labels for different nucleotides. Theassigning of the nucleotide incorporation events may include comparing apoint representing the temporal characteristic and the intensitycharacteristic with the one or more boundaries. The one or more storedcriteria may include centroids of groups of points, each groupcorresponding to a respective nucleotide. The assigning of thenucleotide incorporation events may include determining distancesbetween a point representing the temporal characteristic and theintensity characteristic for an incorporation event to the centroids andassigning the nucleotide incorporation event to a nucleotide with acentroid closest to the point. The stored criteria may be calibrationcriteria stored in non-volatile memory.

Some embodiments relate to a method of identifying nucleotides,including obtaining characteristics of light detected from luminescentlabels associated with the nucleotides during nucleotide incorporationevents. The characteristics may include, for each nucleotideincorporation event: i) a temporal characteristic the light, thetemporal characteristic representing a speed of decay of a probabilityof photon emission by a luminescent label after excitation; and ii) asecond characteristic of the light. The method may further includegrouping points representing the characteristics of the nucleotideincorporation events into groups of points, individual pointsrepresenting at least the temporal characteristic and the intensitycharacteristic for a corresponding nucleotide incorporation event, andassigning the groups of points to individual nucleotides.

Some embodiments relate to a method of calibrating a sequencinginstrument, including obtaining characteristics of light detected fromluminescent labels associated with the nucleotides during nucleotideincorporation events. The characteristics may include, for eachnucleotide incorporation event: i) a temporal characteristic the light,the temporal characteristic representing a speed of decay of aprobability of photon emission by a luminescent label after excitation;and ii) a second characteristic of the light. The method may furtherinclude grouping points representing the characteristics of thenucleotide incorporation events into groups of points, individual pointsrepresenting at least the temporal characteristic and the intensitycharacteristic for a corresponding nucleotide incorporation event,assigning individual groups of points individual nucleotides,determining one or more criteria distinguishing the groups of points,and storing the one or more criteria.

Some embodiments relate to a method of identifying nucleotides,including obtaining characteristics of light detected from luminescentlabels associated with the nucleotides during nucleotide incorporationevents. The characteristics may include, for each nucleotideincorporation event: i) a temporal characteristic the light, thetemporal characteristic representing a speed of decay of a probabilityof photon emission by a luminescent label after excitation; and ii) anintensity characteristic of the light. The method may further includeassigning the nucleotide incorporation events to nucleotides byevaluating the temporal characteristic and the second characteristic inview of stored criteria for a sequencing instrument distinguishingbetween the characteristics of the light for the luminescent labels.

Some embodiments relate to a method including obtaining characteristicsof light detected from luminescent labels associated with thenucleotides during nucleotide incorporation events. The characteristicsmay include, for each nucleotide incorporation event: i) a temporalcharacteristic the light, the temporal characteristic representing aspeed of decay of a probability of photon emission by a luminescentlabel after excitation; and ii) an intensity characteristic of thelight. The method may further include determining one or more criteriadistinguishing groups of points representing the characteristics of thenucleotide incorporation events, individual points representing thetemporal characteristic and the intensity characteristic for acorresponding nucleotide incorporation event.

The method may further include assigning the groups to respectivenucleotides to produce nucleotide assignments for the groups. The methodmay further include assigning the points to nucleotides based on the oneor more criteria and the nucleotide assignments for the groups.

Some embodiments relate to a method including obtaining characteristicsof light detected from luminescent labels associated with thenucleotides during nucleotide incorporation events. The characteristicsmay include, for each nucleotide incorporation event: i) a temporalcharacteristic the light, the temporal characteristic representing aspeed of decay of a probability of photon emission by a luminescentlabel after excitation; and ii) a second characteristic of the light.The method may further include determining one or more criteriadistinguishing groups of points representing the characteristics of thenucleotide incorporation events, individual points representing thetemporal characteristic and the second characteristic for acorresponding nucleotide incorporation event.

Some embodiments relate to a non-transitory computer readable storagemedium having stored thereon instructions, which, when executed by aprocessor, performs any of the methods described herein.

Some embodiments relate to an apparatus including a processor configuredto perform any of the methods described herein.

Some embodiments relate to a sequencing instrument, including aphotodetector configured to receive light from luminescent labels duringa sequencing reaction and a processor configured to perform any of themethods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects and embodiments of the application will be describedwith reference to the following figures. It should be appreciated thatthe figures are not necessarily drawn to scale. Items appearing inmultiple figures are indicated by the same reference number in all thefigures in which they appear.

FIG. 1A is a flowchart of an algorithm implemented by a pulse caller, inaccordance with some embodiments.

FIG. 1B is a plot of probability of photon emission over time afterexcitation and the distribution of the number of photons in time binsdetected by a photodetector, in accordance with some embodiments.

FIG. 1C is a flowchart of a method for determining nucleotideincorporation events using temporal parameter(s) of emitted light, inaccordance with some embodiments.

FIG. 1D is a flowchart of a method for determining nucleotideincorporation events using intensity and temporal characteristic(s) ofemitted light, in accordance with some embodiments

FIG. 2 is a plot of intensity of light detected by a photodetector overtime during sequencing of a nucleic acid, in accordance with someembodiments.

FIG. 2-1A is a block diagram representation of an apparatus that may beused for rapid, mobile analysis of biological and chemical specimens, inaccordance with some embodiments.

FIG. 2-1B is a block diagram of an integrated device and an instrument,in accordance with some embodiments.

FIG. 3 is a flowchart of an algorithm implemented by a base caller, inaccordance with some embodiments.

FIG. 3-1A is a schematic of an integrated device, in accordance withsome embodiments.

FIG. 3-1B is a schematic of excitation energy coupling to sample wellsin a row of pixels and emission energy from each sample well directedtowards sensors, in accordance with some embodiments.

FIG. 4 is a plot of intensity versus time bin ratio as a temporalparameter for nucleotide incorporation events showing clusters of pointsfor different nucleotides, in accordance with some embodiments.

FIG. 5 is a plot showing boundaries and centroid positions for theclusters shown in FIG. 4, in accordance with some embodiments.

FIG. 6 is a flowchart of an algorithm implemented by a base caller toidentify nucleotides based on one or more calibration criteria, inaccordance with some embodiments.

FIG. 7 is a plot of intensity versus temporal parameter illustratingrelative distances of a point corresponding to a nucleotideincorporation event to centroid positions for different nucleotides, inaccordance with some embodiments.

FIG. 8 is a block diagram of an illustrative computing device that maybe used in implementing some embodiments of the technology describedherein.

DETAILED DESCRIPTION

The techniques described herein relate to sequencing of nucleic acids,such as DNA and RNA, and in particular to techniques for identifyingnucleotides based upon data acquired from a sensor. Nucleic acidsequencing allows for the determination of the order and position ofnucleotides in a target nucleic acid. Some nucleic acid sequencingmethods are based on sequencing by synthesis, in which the identity of anucleotide is determined as the nucleotide is incorporated into a newlysynthesized strand of nucleic acid that is complementary to the targetnucleic acid. During sequencing, a polymerizing enzyme (e.g., DNApolymerase) may couple (e.g., attach) to a priming location of a targetnucleic acid molecule and add or incorporate nucleotides to the primervia the action of the polymerizing enzyme, which can be generallyreferred to as a primer extension reaction.

Each nucleotide may be associated with a luminescent molecule (e.g.,fluorophore) that emits light in response to excitation, and which isused to label each type of nucleotide to discriminate among thedifferent types of nucleotides. For example, a set of four labels may beused to label the nucleobases present in DNA such that each marker ofthe set is associated with a different nucleobase, e.g., a first labelbeing associated with adenine (A), a second label being associated withcytosine (C), a third label being associated with guanine (G), and afourth label being associated with thymine (T). A label may be coupledto a nucleotide through bonding of the label to the nucleotide eitherdirectly or indirectly via a linker molecule.

As the primer extension reaction occurs, a nucleotide and its respectiveluminescent labels are retained by the polymerizing enzyme duringincorporation of the nucleotide into the synthesized complementarynucleic acid. The luminescent label can be excited by pulses of lightduring the period in which the nucleotide is incorporated into thesynthesized nucleic acid and emits light characteristic of the label. Insome embodiments, the label is attached, either directly or indirectlythrough a linker molecule, to a terminal phosphate of a nucleotide suchthat the label is detached or released from the nucleotide via theaction of the polymerizing enzyme during incorporation of the nucleotide(e.g., cleavage of a phosphate bond). Sensing and analyzing the lightemitted by the luminescent label in response to the excitation can allowidentifying the nucleotide that was incorporated. As the primerextension reaction occurs, excitation, sensing and analysis is performedfor each subsequent nucleotide added to the synthesized nucleic acid.The sequence of the target nucleic acid can be determined from thecomplementary sequence of the synthesized nucleic acid.

The light emitted by the luminescent label may have a number ofcharacteristics that can be used to distinguish the label from otherlabels, and thus identify a nucleotide. These characteristics includeintensity (e.g., probability of emitting light), a temporalcharacteristic (e.g., rate of decay of the probability of photonemission after excitation, pulse duration for incorporation and/orinterpulse duration before and/or after incorporation), a spectralcharacteristic (e.g., wavelength(s) of light emitted), or anycombination thereof. The light emitted by the luminescent label may bedetected by a photodetector that can detect one of more of thesecharacteristics. An example of a suitable photodetector is described inU.S. patent application Ser. No. 14/821,656 entitled “INTEGRATED DEVICEFOR TEMPORAL BINNING OF RECEIVED PHOTONS,” which is hereby incorporatedby reference in its entirety. As described therein, the photodetectormay have the capability of detecting the arrival times of photons, whichcan allow determining temporal characteristics of the light emitted bythe labels. Detecting temporal characteristics of the emitted light canallow for discriminating between labels that emit light with differenttemporal characteristics. One example of a temporal characteristic isluminance lifetime. A luminescent molecule, such as a fluorophore, mayemit photons in response to excitation. The probability of theluminescent molecule emitting a photon decreases with time after theexcitation occurs. The rate of decay in the probability may beexponential. The “lifetime” is characteristic of how fast theprobability decays over time. A fast decay is said to have a shortlifetime, while a slow decay is said to have a long lifetime. Detectingtemporal characteristics of the light emitted by luminescent moleculescan allow distinguishing luminescent molecules that have differentlifetimes. Labeling different nucleotides with luminescent moleculeshaving different lifetimes can allow distinguishing between thenucleotides based upon a temporal characteristic of the light detected.

The photodetector described in U.S. patent application Ser. No.14/821,656 can detect the time of arrival of photons with nanosecond orpicosecond resolution, and can time-bin the arrival of incident photons.Since the emission of photons is probabilistic, the label may be exciteda plurality of times and any resulting photon emissions may betime-binned. Performing such a measurement a plurality of times allowspopulating a histogram of times at which photons arrived after anexcitation event. This information can be analyzed to calculate atemporal characteristic of the emitted light, which can allowdistinguishing the label from another label based on the temporalcharacteristic.

The techniques described herein can analyze a stream of data from aphotodetector to sequence the nucleic acid based on the characteristicsof the detected light. These techniques may be implemented by a “pulsecaller” and a “base caller,” which may be software and/or hardwaremodules of a sequencing instrument or another device. Generally, a pulsecaller analyzes the stream of data to identify time periods when pulsesof luminescence from the label occur, signifying a dye-conjugatednucleotide being incorporated into the oligonucleotide strand by thepolymerase. A “base caller” analyzes characteristics of the lightdetected during the time periods identified by the pulse caller todetermine, or “call” the identity of the nucleotides.

FIG. 1A shows a flowchart of an algorithm that may be implemented by thepulse caller. In step S1, the intensity of the received light vs. timeis calculated. As discussed above, the photodetector may time bin thearrival of incident photons from a label in response to exposing thelabel to an excitation source (e.g., by a laser pulse). A label may berepeatedly excited, and the arrival of incident photons from the labelmay be time binned. As an example, during a 10 ms measurement period,laser excitation pulses may be emitted at a frequency of 100 MHz toexcite the label. The label may emit a photon with a low probability(e.g., 1 photon emission in 10,000 excitations). If the label is exciteda number of times (e.g., 1 million times) within a 10 ms period,approximately 100 photons may be received. In some instances, a labelmay not become excited after exposure to an excitation source and notemit a photon after an excitation event, which may contribute to the lowprobability of emission. As discussed above, the arrival times of theincident photons with respect to the excitation may be time-binned. Thephotodetector may provide signals representing the number of photons ineach time bin.

FIG. 1B shows an example in which a photodetector time-bins the arrivalof incident photons into eight time bins. Since, as discussed above, theprobability of photon emission decays over time, earlier time bins havemore photons than the later time bins. By repeatedly exciting the labeland detecting the timing of photons emitted, a histogram can bepopulated that approximates the decay in the probability of photonemission over time, as shown in FIG. 1B.

The intensity of the light received over the measurement period (e.g.,10 ms) may be calculated by the pulse caller by summing the valuesrepresenting the number of photons received in each time bin. Forexample, if the photodetector bins the arrival of incident photons intoeight time bins, as shown in FIG. 1B, the number of photons received inthe eight time bins are summed to determine the intensity. However, anynumber of time bins may be used. If the photodetector has two time bins,the values representing the number of photons received in both time binsare summed to determine the intensity. For example, if the first timebin has 100 photons and second time bin has 50 photons, these values maybe summed to determine an intensity of 150 photons. Alternatively, aseparate time bin may exist for the purpose of measuring the totalphoton intensity.

The determination of the intensity of the light received may beperformed for subsequent measurement periods in the data stream from thephotodetector. For example, if the photodetector performs measurementsin 10 ms periods, the intensity may be determined for each measurementperiod by summing the time bins in each 10 ms period. As a result, datarepresenting the intensity of the light received over time can bedetermined.

FIG. 2 shows several minutes of an example trace representing theintensity of the light received vs. time. Because there is significantbaseline and variance in the trace and true pulses often have a lowsignal-to-noise ratio, identifying pulses corresponding to incorporationevents can be challenging. In step S2, a pulse-finding algorithm is runon the intensity vs. time data to identify times when bursts of lightare emitted corresponding to incorporation events.

For the pulse-finding algorithm, one suitable approach is to run achangepoint algorithm on the trace data that determines when shifts inthe mean and variance of the signal occur, e.g., when changing frombackground (i.e., interpulse) to signal (i.e. pulse) and vice versa.After each changepoint is identified, a threshold separates interpulseregions (regions between pulses) from pulse regions on the basis ofchangepoint level (e.g., intensity). This threshold can be determinedmanually, with histogramming, kernel density estimation, or k-meansclustering.

Another suitable approach is to analyze the mean/median and variance ofthe trace, and then define pulses as increases of a certain number ofstandard deviations or more above the mean/median.

Yet another suitable approach is to use a state machine, which is eitherin a pulse or interpulse state, and is judged to alternate between thetwo. Thresholds define transitions between the two states.

In some embodiments, additional filtering of called pulses may occur,such as removing pulses that do not meet a minimum or maximum durationthresholds (as very short pulses and very long pulses are often falsepositives).

The latter two approaches have an additional benefit in that they can beoperated on the data as it is being acquired, whereas the changepointalgorithm may need all of the data in order to operate.

Above is described techniques for identifying pulses corresponding tonucleotide incorporation events based on the intensity of the lightemitted. However, other characteristics of the light emitted may be usedto identify pulses in addition to, or as alternative to intensity. Insome embodiments pulses may be identified based on temporalcharacteristic(s) of the emitted light as an alternative to or inaddition to using intensity. Different nucleotides may be labeled withmolecules that emit light with different temporal characteristics, andthe temporal characteristics may be analyzed to determine whenincorporation events begin and end. As an example, different luminescentlabels may have different “lifetimes” or rates at which the probabilityof photon emission in response to excitation decays over time. A changein the measured lifetime may indicate the start or end of anincorporation event.

FIG. 1C shows a flowchart of a method that uses temporal parameter(s) todetermine when pulses corresponding to incorporation events occur. Instep S3, temporal parameter(s) for the light emitted duringincorporation events is/are determined. For example, as discussed below,temporal characteristics may be determined based upon time bininformation (i.e., information from or based on or more time bins). Insome embodiments, temporal characteristic(s) may be determined by thebase caller and provided to the pulse caller. In step S4, apulse-finding algorithm may be run on data representing the temporalparameter over time. The pulse-finding algorithm may operate similarlyas discussed above with respect to intensity. In some embodiments, bothintensity and temporal characteristic(s) may be used to identify thetimes at which incorporation events occur. As an example, changes in atemporal characteristic may be used to refine the identification ofpulses based on intensity. FIG. 1D shows a flowchart of such a method.In step S1, intensities of light may be obtained for each incorporationevent. The intensities may be calculated by summing the time bins ineach set of time bins, as discussed above. However, the intensities neednot be obtained by summing the time bins, and may be measured and/ordetermined in a different way. In step S2, a pulse-finding algorithm isrun on the intensity vs. time data to identify times when bursts oflight are emitted corresponding to incorporation events. In step S3,temporal parameter(s) for the light emitted during incorporation eventsis/are determined. In step S5, the pulses identified in step S2 may beevaluated and possibly refined based on the temporal parameter(s). Forexample, if a long pulse is identified (e.g., having a length greaterthan a threshold amount), the temporal parameter(s) of the light emittedduring the pulse may be evaluated. If the temporal parameter shiftssignificantly during the pulse (e.g., changes by more than a thresholdamount, or an amount that may indicate a different nucleotide), theinitial pulse-call may be revised to identify two separate pulsesinstead of one long pulse. The time at which the shift in temporalparameter occurs may correspond to a temporal boundary between the twopulses. If the temporal parameter does not shift significantly duringthe pulse (e.g., does not change or changes by a relatively smallamount), the initial pulse-call may be left unchanged. Accordingly, theresults of initial pulse-calling based upon intensity can be evaluatedand/or refined using temporal parameter(s).

In some embodiments, initial pulse-calling may be performed usingtemporal parameter(s), and pulses may be refined using intensityinformation.

As a result of running the pulse-finding algorithm, the pulse calleridentifies the times at which pulses corresponding to incorporationevents occur. For each pulse, the pulse-caller may identify thestart-time and the stop-time, the start-time and the duration, or thestop-time and the duration. The times at which such pulses occur may beanalyzed to identify the luminescent label, and thus its associatednucleotide.

After calling the pulse caller on a stream of data from thephotodetector, the base caller may be called to analyze one or morecharacteristics of the light for each incorporation event. The pulsecaller may pass the times at which pulses occur to the base caller.Optionally the pulse caller may pass additional information to the basecaller, such as the information regarding the number of photons receivedin each time bin, the calculated intensity for each measurement period,or any other suitable information.

FIG. 3 shows a flowchart of an algorithm that may be implemented by thebase caller to identify nucleotides, and/or may be used to calibrate thesequencing instrument.

In step S11, intensities may be obtained for each incorporation event.The intensities may be calculated by summing the time bins in each setof time bins, as discussed above. Alternatively, the base caller mayreceive the intensities from the pulse caller.

An intensity may be normalized for the duration of the incorporationevent identified by the pulse caller. For example, if an incorporationevent lasts for twice as long as a measurement interval, the intensitymay be calculated by summing the time bins for the two measurementintervals and dividing by 2. For instance, if an incorporation eventlast 20 ms, the measurement period is 10 ms, and photons are groupedinto two time bins, the intensity may be calculated by summing thephotons collected in the two time bins of the first measurement as wellas the photons collected in the two time bins of the second measurement,then dividing by two. Such a calculation may also be considered to bethe calculation of an average intensity over the 20 ms incorporationevent.

In step S12, a temporal parameter may be determined for eachincorporation event. The temporal parameter may represent the decay inthe probability of photon emission by a label over time followingexcitation. Any suitable temporal parameter may be used. In someembodiments the luminance lifetime may be calculated by fitting anexponential to the time bins (see e.g., FIG. 1B), and the luminancelifetime may be used as the temporal parameter. In some embodiments, thephoton count for different time bins (or a value representative thereof)may be compared to determine a temporal parameter representing the decayin the probability of photon emission over time. For example, if thearrival of incident photons is binned into two time bins, the ratio ofthe photon count for the two bins may be calculated, and the ratio maybe used as the temporal parameter. In some embodiments, the ratio of thebins may be a proxy for calculating a luminance lifetime. The ratio maybe calculated in any suitable way. In some embodiments, if two time binsare used, the photon count for the time bin closest in time to theexcitation event may be divided by the photon count for the second timebin to produce the ratio. In some embodiments, the photon count of thetime bins or the value representative thereof may be normalized (e.g.,by the summed intensity over a set of time bins), and the normalizedvalues may be used to determine the temporal parameter. In someembodiments, the time bin with the maximum photon count may be used asthe temporal parameter. To determine the time bin with the maximumphoton count, the photon counts for the time bins may be compared withone another. As an example with two time bins, the photon count for afirst time bin may be compared with the photon count for a second timebin. The bin with the higher photon count may be selected as a temporalparameter, and may be used for discriminating luminescent molecules. Forexample, one luminescent molecule may have a relatively short lifetime,which may result in the first time bin (closest in time to theexcitation event) having the maximum photon count, and anotherluminescent molecule may have a relatively long lifetime, which mayresult in another time bin (farther in time from the excitation event)having the maximum photon count.

Although FIG. 3 shows step S11 as being performed prior to step S12,this is merely by way of illustration, as step S12 may be performedprior to step S11, or steps 511 and S12 may be performed concurrently.

FIG. 4 shows that the intensity and temporal parameter for eachincorporation event may be plotted as a point in two-dimensional space,with intensity and temporal parameter being on respective axes. In thisexample, the temporal parameter is plotted on the horizontal (x) axisand intensity is plotted on the vertical (y) axis. Four different labelsmay be used for the nucleotides that can be distinguished from oneanother based upon the intensity, the temporal parameter, or both. Asshown in FIG. 4, plotting the measured intensity and temporal parameterfor each incorporation event results in four clusters of pointscorresponding to the four nucleotides A, C, G and T.

In step S13, the points may be assigned to groups (also referred toherein as “clusters”). In some embodiments, a clustering algorithm maybe run on the points to assign the points for each incorporation eventto one of four clusters. For example, the clustering algorithm mayperform k-means clustering of the pulses in n-dimensional space, where kis 4 (A, C, G, T), and n is the number of metrics being used forbase-calling. However, in some embodiments more than four clusters maybe assigned. If more than four clusters are assigned, clustering may beperformed in which k is greater than 4. The inventors have recognizedand appreciated that in some cases the clusters may not bewell-resolved, and it may be advantageous to group the points into morethan four clusters. In such a case, more than one cluster may beassigned to the same nucleotide. In some embodiments, filtering may beperformed to eliminate points that are outliers. For example, if a pointhas a temporal parameter and/or intensity that is outside of an expectedrange, it may be excluded from the clustering algorithm and/or may notbe assigned to any nucleotide group.

Any suitable number of points may be provided to the clusteringalgorithm, such as greater than 50, greater than 100, greater than 500,etc. The result of the clustering algorithm is to group each point intoone of the four (or more) clusters. In the example of FIG. 4, n=2because two metrics, intensity and temporal parameter, are used. Atwo-dimensional example with intensity and time bin ratio as a temporalparameter is plotted in FIG. 4. However, other metrics may be used.

Another two-dimensional example involves obtaining both a temporalparameter and a spectral parameter, with the spectral parameter being onthe vertical (y) axis of FIG. 4 rather than intensity. In this example,spectral information is obtained regarding the light emitted for eachincorporation event, and used for distinguishing the nucleotides.

However, any number of metrics may be used, not limited to two. Forexample, in some embodiments spectral information for an incorporationevent may be obtained in addition to intensity and a temporal parameter,which can be plotted as points in three-dimensional space, withintensity, temporal parameter, and spectral information being onrespective axes.

After grouping the points, it may be beneficial to further refine thegroups, potentially with more metrics than were used in the initialgrouping step. For this purpose a support vector machine (SVM) or othersupervised classifier can be used. Clustering labels may be used asinitial training data. This process may be repeated, using the resultsfrom the most recent iteration of the classifier as the training for thenext iteration, until it converges.

Although a clustering algorithm may be used to assign points toclusters, in some embodiments the points may be assigned to groupswithout using a clustering algorithm. In some embodiments, boundariesbetween groups of points may be determined without running a clusteringalgorithm.

In step S14, the clusters of points may be assigned to nucleotides. Thisassignment may be performed based on known characteristics of thelabels. For example, in the plot of FIG. 4, it may be known that thelabel for a T has a high intensity and the lowest lifetime, the labelfor A has a low intensity and a moderate lifetime, the label for G has ahigh intensity and a moderate lifetime, and the label for C has thehighest lifetime and a high intensity. The clusters of points may beassigned to bases using the position of the clusters relative to oneanother. For example, the cluster with the lowest lifetime may beassigned to T, the cluster with the highest lifetime may be assigned toC, the cluster with the lowest intensity may be assigned to A, and theremaining cluster may be assigned to G. The points in each cluster maybe assigned the nucleotide of their cluster. By storing informationabout the time each measurement of intensity and temporal characteristicwas performed, the nucleotide strand can be sequenced.

If the method is used to perform sequencing, the method may terminate atthis point. If the method is used for calibration, the method maycontinue to step S15. The inventors have recognized and appreciated thatif an initial calibration is performed, it is not necessary to run aclustering algorithm to assign all the points to nucleotides. In someembodiments, calibration criteria may be determined for assigning apoint to a nucleotide type. As an example, following the clustering instep S13 or the assigning of the nucleotides in step S14, boundariesbetween the different types of nucleotides may be determined. Theboundaries may be functions defining regions of a phase space asillustrated in FIG. 5. Axes of the phase space may include intensity,temporal parameter, emission wavelength, and/or excitation wavelength ofthe excitation laser pulses. As an example, line segments or curves intwo-dimensional space may be selected that delineate the boundaries 51between the different nucleotides, as shown in FIG. 5. Inhigher-dimensional space, the boundaries may be surfaces orhigher-dimensional objects (termed “hyperplanes”). Once boundaries 51are determined, the points can be assigned to nucleotides by evaluatingtheir positions with respect to the boundaries, and clustering need notbe performed. Accordingly, in some embodiments, a sequencing instrumentmay be calibrated to delineate the boundaries 51. The calibrationprocess may be performed using the same set of labels as duringsequencing of a nucleic acid. As another example of performingcalibration in step S15, the centroids of the clusters may bedetermined, which can allow assigning the points to nucleotides based onwhich cluster has a centroid that is closest to an individual point.Regardless of the type of calibration criteria that are determined, thecalibration criteria are then stored (e.g., in a memory of theinstrument) for later use.

Calibration may be performed at any suitable time. In some embodiments,calibration may be desirable prior to first using the instrument, uponusing a new set of labels, upon a change in environmental conditions inwhich the instrument is used, or after a period of use to account foraging of components of the instrument. The calibration may be performedin response to a request from a user, such as by pressing a button onthe instrument or sending a calibration command to the instrument fromanother device, or automatically based on a schedule or on an as-neededbasis in response to the instrument software determining the performanceis sub-optimal. Once the calibration criteria are obtained, sequencingcan be performed more quickly by evaluating the detected points withrespect to the calibration criteria.

FIG. 6 shows a flowchart of an algorithm that may be used by the basecaller to identify nucleotides based on one or more calibrationcriteria. The parameters of the light (e.g., intensity and a temporalparameter) may be determined in steps S11 and S12, which may be the sameas those shown in FIG. 3, and steps S11 and S12 may be performed in anyorder, as discussed above. In step S33, nucleotides may be identified byevaluating the measured parameters of the light (e.g., intensity andtemporal parameter) using the stored calibration information. Forexample, if the stored calibration information includes one or moreboundaries between nucleotide clusters, the points can be assigned tonucleotides by comparing the points to the boundaries, which is morecomputationally efficient than performing clustering. As anotherexample, the points can be assigned to nucleotides by calculating thedistance of a point to each of the four centroids of the nucleotideclusters, then assigning the point to the nucleotide with the centroidthat is the closest. This technique is illustrated in FIG. 7, whichshows a point 61 representing a measured intensity and temporalparameter. Also shown in FIG. 6 are the centroids for the labelscorresponding to the four nucleotides. To determine which centroid iscloset, the distance from point 61 to each of the four centroids may becalculated, and the nucleotide is assigned to the point 61 which has itscentroid the shortest distance from the point 61. As shown, point 61 isclosest to the centroid for the label corresponding to the nucleotide“A.” Accordingly, point 61 is determined to correspond to the nucleotide“A.”

In some embodiments, identifying nucleotides includes performingclustering on a first portion of points associated with incorporationevents and using calibration criteria to perform basecalls on a secondportion of points. The first portion may include any suitable number ofpoints to provide a desired level of accuracy in the calibrationcriteria.

In some embodiments a confidence level that a point corresponds to aparticular type of nucleotide may be determined. As an example, thedistance of a point from a centroid of a region, such as the centroidsshown in FIG. 5, may be used to determine a confidence level for thepoint. Points having a small distance to the centroid may have a highconfidence level indicating that the point is very likely correctlyidentified as corresponding to a nucleotide, while points having alarger distance from the centroid, or which are barely closer to onecentroid than another are less likely to be correctly identified. Inthis example, the confidence level may be quantified based upon thedistance between the point and the centroid, or based on comparing thedistance between the point and the centroid with the distance betweenthe point and one or more other centroids. As another example, if thecalibration criteria includes one or more boundaries between clusters,the confidence level may be quantified by determining the distancebetween the point and one or more boundaries. Points that are closer toa boundary may be given a lower confidence level. In some embodiments,the confidence level for each nucleotide identification may be stored inaddition to storing the nucleotide identification itself.

In some embodiments, the confidence level depends on the calibrationcriteria and how well the calibration criteria fit the calibration data.The more accurately the calibration criteria fit the calibration data,the higher the confidence levels may be for different points.

In some embodiments, the confidence level may depend on the timeduration of the incorporation event associated with a point because theconfidence level can depend on the signal-to-noise ratio of the pulseidentified by the pulse caller. As an example, a long time duration mayindicate that the pulse caller failed to identify two subsequentincorporation events, such as incorporation events of the samenucleotide type. In some embodiments, the base caller may communicatewith the pulse caller to request that the pulse caller reevaluate thetime duration of the incorporation event.

In some embodiments, previously-derived boundaries (e.g., an SVM model)may be applied to new pulse calls to determine the appropriatenucleotide incorporated at each pulse call event. Pulse call metrics arefirst scaled, then, the previously derived boundaries can be applied toclassify that incorporation event.

In order to derive boundaries that generalize across the pulse call datafrom multiple pixels, it may be necessary to scale (or normalize) eachset of pulse call data from each pixel in the array prior to includingthose data in the calibration dataset. By scaling the intensity metric,by clustering only on intensity, and using one or more of those clustersas the mean or median of intensity, one can normalize the intensitymetric of all incoming pulse calls. This scaling, or normalization, isapplied both during the calibration phase, as well as during the basecalling phase using the stored calibration data. This has the benefit ofnot requiring boundaries be generated for each pixel in the array, whichis a performance improvement, and enables scaling to very large arrayswhere all the data may not typically fit into RAM at once. A furtherbenefit is a reduction in runtime, since a smaller number of pulseswould need to be separated by intensity and scaled or normalized to thecalibration data set. This approach also allows for fewer pulses to bestored and grouped, prior to establishing the scaling or normalizationfactors, thus allowing outputting base calls in near real time as thedata is acquired from the pixel array.

Having described techniques that may be implemented by a pulse callerand a base caller to perform sequencing and/or calibration of asequencing instrument, an example of a suitable sequencing instrumentwill now be described. In some embodiments, the instrument is configuredto interface with an integrated device that includes an array of pixels.A surface of the integrated device has a plurality of sample wells,where a sample well is configured to receive a sample from a specimenplaced on the surface of the integrated device. A specimen may containmultiple samples, and in some embodiments, different types of samples.The plurality of sample wells may have a suitable size and shape suchthat at least a portion of the sample wells receive one sample from aspecimen. In some embodiments, the number of samples within a samplewell may be distributed among the sample wells such that some samplewells contain one sample with others contain zero, two or more samples.

In some embodiments, a specimen may contain multiple single-stranded DNAtemplates, and individual sample wells on a surface of an integrateddevice may be sized and shaped to receive a single-stranded DNAtemplate. Single-stranded DNA templates may be distributed among thesample wells of the integrated device such that at least a portion ofthe sample wells of the integrated device contain a single-stranded DNAtemplate. The specimen may also contain tagged dNTPs which then enter inthe sample well and may allow for identification of a nucleotide as itis incorporated into a strand of DNA complementary to thesingle-stranded DNA template in the sample well. In such an example, the“sample” may refer to both the single-stranded DNA and the tagged dNTPcurrently being incorporated by a polymerase. In some embodiments, thespecimen may contain single-stranded DNA templates and tagged dNTPS maybe subsequently introduced to a sample well as nucleotides areincorporated into a complementary strand of DNA within the sample well.In this manner, timing of incorporation of nucleotides may be controlledby when tagged dNTPs are introduced to the sample wells of an integrateddevice.

Excitation energy is provided from an excitation source located separatefrom the pixel array of the integrated device. The excitation energy isdirected at least in part by elements of the integrated device towardsone or more pixels to illuminate an illumination region within thesample well. A label may then emit emission energy when located withinthe illumination region and in response to being illuminated byexcitation energy. In some embodiments, one or more excitation sourcesare part of the instrument of the system where components of theinstrument and the integrated device are configured to direct theexcitation energy towards one or more pixels.

Emission energy emitted by a sample may then be detected by one or moresensors within a pixel of the integrated device. Characteristics of thedetected emission energy may provide an indication for identifying themarked associated with the emission energy. Such characteristics mayinclude any suitable type of characteristic, including an arrival timeof photons detected by a sensor, an amount of photons accumulated overtime by a sensor, and/or a distribution of photons across two or moresensors. In some embodiments, a sensor may have a configuration thatallows for the detection of one or more timing characteristicsassociated with a sample's emission energy (e.g., fluorescencelifetime). The sensor may detect a distribution of photon arrival timesafter a pulse of excitation energy propagates through the integrateddevice, and the distribution of arrival times may provide an indicationof a timing characteristic of the sample's emission energy (e.g., aproxy for fluorescence lifetime). In some embodiments, the one or moresensors provide an indication of the probability of emission energyemitted by the label (e.g., fluorescence intensity). In someembodiments, a plurality of sensors may be sized and arranged to capturea spatial distribution of the emission energy. Output signals from theone or more sensors may then be used to distinguish a label from among aplurality of labels, where the plurality of labels may be used toidentify a sample within the specimen.

A schematic overview of the system 2-100 is illustrated in FIGS. 2-1Aand 2-1B. The system comprises an integrated device 2-102 thatinterfaces with an instrument 2-104. In some embodiments, instrument2-104 may include one or more excitation sources 2-106 integrated aspart of instrument 2-104. In some embodiments, an excitation source maybe external to both instrument 2-104 and integrated device 2-102, andinstrument 2-104 may be configured to receive excitation energy from theexcitation source and direct it to the integrated device. The integrateddevice may interface with the instrument using any suitable socket forreceiving the integrated device and holding it in precise opticalalignment with the excitation source. The excitation source 2-106 may beconfigured to provide excitation energy to the integrated device 2-102.As illustrated schematically in FIG. 2-1B, the integrated device 2-102has multiple pixels, where at least a portion of pixels 2-112 mayperform independent analysis of a sample. Such pixels 2-112 may bereferred to as “passive source pixels” since a pixel receives excitationenergy from a source 2-106 separate from the pixel, where the sourceexcites a plurality of pixels. A pixel 2-112 has a sample well 2-108configured to receive a sample and a sensor 2-110 for detecting emissionenergy emitted by the sample in response to illuminating the sample withexcitation energy provided by the excitation source 2-106. Sample well2-108 may retain the sample in proximity to a surface of integrateddevice 2-102 to provide ease in delivery of excitation energy to thesample and detection of emission energy from the sample.

Optical elements for guiding and coupling excitation energy to thesample well 2-108 are located both on integrated device 2-102 and theinstrument 2-104. Such source-to-well elements may comprise one or moregrating couplers located on integrated device 2-102 to couple excitationenergy to the integrated device and waveguides to deliver excitationenergy from instrument 2-104 to sample wells in pixels 2-112. In someembodiments, elements located on the integrated device may act to directemission energy from the sample well towards the sensor. Sample well2-108, a portion of the excitation source-to-well optics, and the samplewell-to-sensor optics are located on integrated device 2-102. Excitationsource 2-106 and a portion of the source-to-well components are locatedin instrument 2-104. In some embodiments, a single component may play arole in both coupling excitation energy to sample well 2-108 anddelivering emission energy from sample well 2-108 to sensor 2-110.Examples of suitable components, for coupling excitation energy to asample well and/or directing emission energy to a sensor, to include inan integrated device are described in U.S. patent application Ser. No.14/821,688 entitled “INTEGRATED DEVICE FOR PROBING, DETECTING ANDANALYZING MOLECULES,” and U.S. patent application Ser. No. 14/543,865entitled “INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING,DETECTING, AND ANALYZING MOLECULES,” both of which are incorporated byreference in their entirety.

As illustrated in FIG. 2-1B, the integrated device comprises a pluralityof pixels where a pixel 2-112 is associated with its own individualsample well 2-108 and at least one sensor 2-110. The plurality of pixelsmay be arranged in an array, and there may be any suitable number ofpixels in the array. The number of pixels in integrated device 2-102 maybe in the range of approximately 10,000 pixels to 1,000,000 pixels orany value or range of values within that range. In some embodiments, thepixels may be arranged in an array of 512 pixels by 512 pixels.Integrated device 2-102 and instrument 2-104 may include multi-channel,high-speed communication links for handling data associated with largepixel arrays (e.g., more than 10,000 pixels).

Instrument 2-104 interfaces with integrated device 2-102 throughintegrated device interface 2-114. Integrated device interface 2-114 mayinclude components to position and/or align integrated device 2-102 toinstrument 2-104 to improve coupling of excitation energy fromexcitation source 2-106 to integrated device 2-102. Excitation source2-106 may be any suitable light source that is arranged to deliverexcitation energy to at least one sample well. Examples of suitableexcitation sources are described in U.S. patent application Ser. No.14/821,688 entitled “INTEGRATED DEVICE FOR PROBING, DETECTING ANDANALYZING MOLECULES,” which is incorporated by reference in itsentirety. In some embodiments, excitation source 2-106 includes multipleexcitation sources that are combined to deliver excitation energy tointegrated device 2-102. The multiple excitation sources may beconfigured to produce multiple excitation energies or wavelengths. Theintegrated device interface 2-114 may receive readout signals from thesensors in the pixels located on the integrated device. The integrateddevice interface 2-114 may be designed such that the integrated deviceattaches to the instrument by securing the integrated device to theintegrated device interface 2-114.

The instrument 2-104 includes a user interface 2-116 for controlling theoperation of instrument 2-104. The user interface 2-116 is configured toallow a user to input information into the instrument, such as commandsand/or settings used to control the functioning of the instrument. Insome embodiments, the user interface 2-116 may include buttons,switches, dials, and a microphone for voice commands. Additionally, theuser interface 2-116 may allow a user to receive feedback on theperformance of the instrument and/or integrated device, such as properalignment and/or information obtained by readout signals from thesensors on the integrated device. In some embodiments, the userinterface 2-116 may provide feedback using a speaker to provide audiblefeedback, and indicator lights and/or display screen for providingvisual feedback. In some embodiments, the instrument 2-104 includes acomputer interface 2-118 used to connect with a computing device 2-120.Any suitable computer interface 2-118 and computing device 2-120 may beused. For example, the computer interface 2-118 may be a USB interfaceor a FireWire interface. The computing device 2-120 may be any generalpurpose computer, such as a laptop or desktop computer. The computerinterface 2-118 facilitates communication of information between theinstrument 2-104 and the computing device 2-120. Input information forcontrolling and/or configuring the instrument 2-104 may be providedthrough the computing device 2-120 connected to the computer interface2-118 of the instrument. Output information may be received by thecomputing device 2-120 through the computer interface 2-118. Such outputinformation may include feedback about performance of the instrument2-104 and/or integrated device 2-112 and information from the readoutsignals of the sensor 2-110. The instrument 2-104 may also include aprocessing device 2-122 for analyzing data received from the sensor2-110 and/or sending control signals to the excitation source 2-106. Insome embodiments, the processing device 2-122 may comprise a generalpurpose processor, a specially-adapted processor (e.g., a centralprocessing unit (CPU) such as one or more microprocessor ormicrocontroller cores, a field-programmable gate array (FPGA), anapplication-specific integrated circuit (ASIC), a custom integratedcircuit, a digital signal processor (DSP), or a combination thereof.) Insome embodiments, the processing of data from the sensor 2-110 may beperformed by both the processing device 2-122 and the external computingdevice 2-120. In other embodiments, the computing device 2-120 may beomitted and processing of data from the sensor 2-110 may be performedsolely by processing device 2-122.

A cross-sectional schematic of the integrated device 3-102 illustratinga row of pixels is shown in FIG. 3-1A. Each pixel 3-112 includes asample well 3-108 and a sensor 3-110. The sensor 3-110 may be alignedand positioned to sample well 3-112 such that sensor 3-110 receivesemission energy emitted by a sample within sample well 3-112. Examplesof suitable sensors are described in U.S. patent application Ser. No.14/821,656 entitled “INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVEDPHOTONS,” which is incorporated by reference in its entirety.

An excitation source coupled to the integrated device may provideexcitation energy to one or more pixels of integrated device 3-102. FIG.3-1B is a schematic illustrating coupling of excitation source 3-106 tointegrated device 3-102 to provide excitation energy 3-130 (shown indashed lines) to integrated device 3-102. FIG. 3-1B illustrates the pathof excitation energy from excitation energy source 3-106 to a samplewell 3-108 in pixel 3-112. Components located off of the integrateddevice may be used to position and align the excitation source 3-106 tothe integrated device. Such components may include optical componentsincluding lenses, mirrors, prisms, apertures, attenuators, and/oroptical fibers. Additional mechanical components may be included in theinstrument to allow for control of one or more alignment components.Such mechanical components may include actuators, stepper motors, and/orknobs.

The integrated device includes components that direct the excitationenergy 3-130 towards pixels in the integrated device. Within each pixel3-112, excitation energy is coupled to the sample well 3-108 associatedwith the pixel. Although FIG. 3-1B illustrates excitation energycoupling to each sample well in a row of pixels, in some embodiments,excitation energy may not couple to all of the pixels in a row. In someembodiments, excitation energy may couple to a portion of pixels orsample wells in a row of pixels of the integrated device. Excitationenergy may illuminate a sample located within a sample well. The samplemay reach an excited state in response to being illuminated by theexcitation energy. When a sample is in an excited state, the sample mayemit emission energy and the emission energy may be detected by asensor. FIG. 3-1B schematically illustrates the path of emission energy3-140 (shown as solid lines) from sample well 3-108 to sensor 3-110 ofpixel 3-112. Sensor 3-110 in pixel 3-112 may be configured andpositioned to detect emission energy from sample well 3-108. In someembodiments, sensor 3-110 may include multiple sub-sensors.

A sample to be analyzed may be introduced into sample well 3-108 ofpixel 3-112. The sample may be a biological sample or any other suitablesample, such as a chemical sample. The sample may include multiplemolecules and the sample well may be configured to isolate a singlemolecule. In some instances, the dimensions of the sample well may actto confine a single molecule within the sample well, allowingmeasurements to be performed on the single molecule. An excitationsource 3-106 may be configured to deliver excitation energy into thesample well 3-108, so as to excite the sample or at least oneluminescent marker attached to the sample or otherwise associated withthe sample while it is within an illumination area within the samplewell 3-108.

When an excitation source delivers excitation energy to a sample well,at least one sample within the well may luminesce, and the resultingemission may be detected by a sensor. As used herein, the phrases “asample may luminesce” or “a sample may emit radiation” or “emission froma sample” mean that a luminescent tag, marker, or reporter, the sampleitself, or a reaction product associated with the sample may produce theemitted radiation.

One or more components of an integrated device may direct emissionenergy towards a sensor. The emission energy or energies may be detectedby the sensor and converted to at least one electrical signal. Theelectrical signals may be transmitted along conducting lines in thecircuitry of the integrated device connected to the instrument throughthe integrated device interface, such as integrated device interface2-114 of instrument 2-104 shown in FIG. 2-1B. The electrical signals maybe subsequently processed and/or analyzed. Processing or analyzing ofelectrical signals may occur on a suitable computing device eitherlocated on the instrument 2-104 or off instrument, such as computingdevice 2-120 shown in FIG. 2-1B.

In operation, parallel analyses of samples within the sample wells arecarried out by exciting the samples within the wells using theexcitation source and detecting signals from sample emission with thesensors. Emission energy from a sample may be detected by acorresponding sensor and converted to at least one electrical signal.The resulting signal, or signals, may be processed on the integrateddevice in some embodiments, or transmitted to the instrument forprocessing by the processing device and/or computing device. Signalsfrom a sample well may be received and processed independently fromsignals associated with the other pixels.

In some embodiments, a sample may be labeled with one or more markers,and emission associated with the markers is discernable by theinstrument. For example the sensor may be configured to convert photonsfrom the emission energy into electrons to form an electrical signalthat may be used to discern a lifetime that is dependent on the emissionenergy from a specific marker. By using markers with different lifetimesto label samples, specific samples may be identified based on theresulting electrical signal detected by the sensor.

A sample may contain multiple types of molecules and differentluminescent markers may uniquely associate with a molecule type. Duringor after excitation, the luminescent marker may emit emission energy.One or more properties of the emission energy may be used to identifyone or more types of molecules in the sample. Properties of the emissionenergy used to distinguish among types of molecules may include afluorescence lifetime value, intensity, and/or emission wavelength. Asensor may detect photons, including photons of emission energy, andprovide electrical signals indicative of one or more of theseproperties. In some embodiments, electrical signals from a sensor mayprovide information about a distribution of photon arrival times acrossone or more time intervals. The distribution of photon arrival times maycorrespond to when a photon is detected after a pulse of excitationenergy is emitted by an excitation source. A value for a time intervalmay correspond to a number of photons detected during the time interval.Relative values across multiple time intervals may provide an indicationof a temporal characteristic of the emission energy (e.g., lifetime).Analyzing a sample may include distinguishing among markers by comparingvalues for two or more different time intervals within a distribution.In some embodiments, an indication of the intensity may be provided bydetermining a number of photons across all time bins in a distribution.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenosine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), or variants thereof. In some examples,a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),or derivatives thereof. A nucleic acid may be single-stranded or doublestranded. A nucleic acid may be circular.

The term “nucleotide,” as used herein, generally refers to a nucleicacid subunit, which can include A, C, G, T or U, or variants or analogsthereof. A nucleotide can include any subunit that can be incorporatedinto a growing nucleic acid strand. Such subunit can be an A, C, G, T,or U, or any other subunit that is specific to one or more complementaryA, C, G, T or U, or complementary to a purine (i.e., A or G, or variantor analogs thereof) or a pyrimidine (i.e., C, T or U, or variant oranalogs thereof).

A nucleotide generally includes a nucleoside and at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more phosphate (PO₃) groups. A nucleotide can includea nucleobase, a five-carbon sugar (either ribose or deoxyribose), andone or more phosphate groups. Ribonucleotides are nucleotides in whichthe sugar is ribose. Deoxyribonucleotides are nucleotides in which thesugar is deoxyribose. A nucleotide can be a nucleoside monophosphate ora nucleoside polyphosphate. A nucleotide can be a deoxyribonucleosidepolyphosphate, such as, e.g., a deoxyribonucleoside triphosphate, whichcan be selected from deoxyadenosine triphosphate (dATP), deoxycytidinetriphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridinetriphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, thatinclude detectable labels (e.g., fluorophores).

In some embodiments, techniques described herein may be carried outusing one or more computing devices. Embodiments are not limited tooperating with any particular type of computing device.

FIG. 8 is a block diagram of an illustrative computing device 1000.Computing device 1000 may include one or more processors 1001 and one ormore tangible, non-transitory computer-readable storage media (e.g.,memory 1003). Memory 1003 may store, in a tangible non-transitorycomputer-recordable medium, computer program instructions that, whenexecuted, implement any of the above-described functionality.Processor(s) 1001 may be coupled to memory 1003 and may execute suchcomputer program instructions to cause the functionality to be realizedand performed.

Computing device 1000 may also include a network input/output (I/O)interface 1005 via which the computing device may communicate with othercomputing devices (e.g., over a network), and may also include one ormore user I/O interfaces 1007, via which the computing device mayprovide output to and receive input from a user. The user I/O interfacesmay include devices such as a keyboard, a mouse, a microphone, a displaydevice (e.g., a monitor or touch screen), speakers, a camera, and/orvarious other types of I/O devices.

The above-described embodiments can be implemented in any of numerousways. For example, the embodiments may be implemented using hardware,software or a combination thereof. When implemented in software, thesoftware code can be executed on any suitable processor (e.g., amicroprocessor) or collection of processors, whether provided in asingle computing device or distributed among multiple computing devices.It should be appreciated that any component or collection of componentsthat perform the functions described above can be generically consideredas one or more controllers that control the above-discussed functions.The one or more controllers can be implemented in numerous ways, such aswith dedicated hardware, or with general purpose hardware (e.g., one ormore processors) that is programmed using microcode or software toperform the functions recited above.

In this respect, it should be appreciated that one implementation of theembodiments described herein comprises at least one computer-readablestorage medium (e.g., RAM, ROM, EEPROM, flash memory or other memorytechnology, CD-ROM, digital versatile disks (DVD) or other optical diskstorage, magnetic cassettes, magnetic tape, magnetic disk storage orother magnetic storage devices, or other tangible, non-transitorycomputer-readable storage medium) encoded with a computer program (i.e.,a plurality of executable instructions) that, when executed on one ormore processors, performs the above-discussed functions of one or moreembodiments. The computer-readable medium may be transportable such thatthe program stored thereon can be loaded onto any computing device toimplement aspects of the techniques discussed herein. In addition, itshould be appreciated that the reference to a computer program which,when executed, performs any of the above-discussed functions, is notlimited to an application program running on a host computer. Rather,the terms computer program and software are used herein in a genericsense to reference any type of computer code (e.g., applicationsoftware, firmware, microcode, or any other form of computerinstruction) that can be employed to program one or more processors toimplement aspects of the techniques discussed herein.

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Also, the invention may be embodied as a method, of which an example hasbeen provided. The acts performed as part of the method may be orderedin any suitable way. Accordingly, embodiments may be constructed inwhich acts are performed in an order different than illustrated, whichmay include performing some acts simultaneously, even though shown assequential acts in illustrative embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

What is claimed is: 1.-35. (canceled)
 36. A sequencing method,comprising: obtaining, by a processor, characteristics of light receivedby a photodetector from luminescent labels during nucleotideincorporation events of a sequencing reaction, the luminescent labelsbeing associated with nucleotides, the characteristics including, forindividual nucleotide incorporation events, a temporal characteristic ofthe light, the temporal characteristic representing a speed of decay ofa probability of photon emission by a luminescent label afterexcitation; and an intensity characteristic of the light, wherein thetemporal characteristic and the intensity characteristic arecharacteristics of light received from a luminescent label during anucleotide incorporation event; and wherein the temporal characteristicand the intensity characteristic are used to perform one or more of:identifying individual nucleotides, and calibrating a sequencinginstrument, and wherein the intensity characteristic represents aquantity of photogenerated charge carriers produced over time by thephotodetector from the light received from the luminescent label duringthe nucleotide incorporation event.
 37. The sequencing method of claim36, wherein the temporal characteristic and the intensity characteristicare used to perform identifying individual nucleotides, and wherein theidentifying of the individual nucleotides comprises: grouping pointsrepresenting the characteristics into groups of points, individualpoints representing at least the temporal characteristic and theintensity characteristic for a corresponding nucleotide incorporationevent; and assigning the groups of points to individual nucleotides. 38.The sequencing method of claim 37, wherein the points are grouped usinga clustering algorithm.
 39. The sequencing method of claim 38, whereinthe clustering algorithm performs k-means clustering in which k isgreater than or equal to four.
 40. The sequencing method of claim 38,wherein the groups of points are assigned to the individual nucleotidesbased on predetermined light-emitting characteristics of the luminescentlabels.
 41. The sequencing method of claim 37, wherein the temporalcharacteristic and the intensity characteristic are used to performidentifying individual nucleotides, and wherein the identifying of theindividual nucleotides comprises: determining one or more criteriadistinguishing groups of points representing the characteristics,individual points representing the temporal characteristic and theintensity characteristic for a corresponding nucleotide incorporationevent; assigning the groups to respective nucleotides to producenucleotide assignments for the groups; and assigning the points tonucleotides based on the one or more criteria and the nucleotideassignments for the groups.
 42. The sequencing method of claim 36,wherein the temporal characteristic and the intensity characteristic areused to perform identifying individual nucleotides, wherein theidentifying of the individual nucleotides comprises assigning thenucleotide incorporation events to nucleotides by evaluating thetemporal characteristic and the intensity characteristic in view ofstored criteria for the sequencing instrument, and distinguishingbetween the characteristics of the light for the luminescent labels. 43.The sequencing method of claim 42, wherein the stored criteria comprisesone or more boundaries between characteristics of the luminescent labelsfor different nucleotides, and the assigning of the nucleotideincorporation events comprises comparing a point representing thetemporal characteristic and the intensity characteristic with the one ormore boundaries.
 44. The sequencing method of claim 42, wherein thestored criteria comprises centroids of groups of points, each groupcorresponding to a respective nucleotide, and the assigning of thenucleotide incorporation events comprises: determining distances betweena point representing the temporal characteristic and the intensitycharacteristic for an incorporation event to the centroids; andassigning the nucleotide incorporation event to a nucleotide with acentroid closest to the point.
 45. The sequencing method of claim 37,wherein the temporal characteristic and the intensity characteristic areused to perform identifying of individual nucleotides, wherein theidentifying the individual nucleotides comprises: determining one ormore criteria distinguishing groups of points representing thecharacteristics of the nucleotide incorporation events, individualpoints representing the temporal characteristic and the intensitycharacteristic for a corresponding nucleotide incorporation event;assigning the groups to respective nucleotides to produce nucleotideassignments for the groups; and assigning the points to nucleotidesbased on the one or more criteria and the nucleotide assignments for thegroups.
 46. A non-transitory computer readable storage medium havingstored thereon instructions, which, when executed by a processor,performs a sequencing method, comprising: obtaining, by a processor,characteristics of light received by a photodetector from luminescentlabels during nucleotide incorporation events of a sequencing reaction,the luminescent labels being associated with nucleotides, thecharacteristics including, for individual nucleotide incorporationevents, a temporal characteristic of the light, the temporalcharacteristic representing a speed of decay of a probability of photonemission by a luminescent label after excitation; and an intensitycharacteristic of the light, wherein the temporal characteristic and theintensity characteristic are characteristics of light received from aluminescent label during a nucleotide incorporation event; and whereinthe temporal characteristic and the intensity characteristic are used toperform one or more of: identifying individual nucleotides, andcalibrating a sequencing instrument, and wherein the intensitycharacteristic represents a quantity of photogenerated charge carriersproduced over time by the photodetector from the light received from theluminescent label during the nucleotide incorporation event.
 47. Thenon-transitory computer readable storage medium of claim 46, wherein thetemporal characteristic and the intensity characteristic are used toperform identifying individual nucleotides, and wherein the identifyingof the individual nucleotides comprises: grouping points representingthe characteristics into groups of points, individual pointsrepresenting at least the temporal characteristic and the intensitycharacteristic for a corresponding nucleotide incorporation event; andassigning the groups of points to individual nucleotides.
 48. Thenon-transitory computer readable storage medium of claim 47, wherein thepoints are grouped using a clustering algorithm.
 49. The non-transitorycomputer readable storage medium of claim 48, wherein the clusteringalgorithm performs k-means clustering in which k is greater than orequal to four.
 50. The non-transitory computer readable storage mediumof claim 48, wherein the groups of points are assigned to the individualnucleotides based on predetermined light-emitting characteristics of theluminescent labels.
 51. The non-transitory computer readable storagemedium of claim 47, wherein the temporal characteristic and theintensity characteristic are used to perform identifying individualnucleotides, and wherein the identifying of the individual nucleotidescomprises: determining one or more criteria distinguishing groups ofpoints representing the characteristics, individual points representingthe temporal characteristic and the intensity characteristic for acorresponding nucleotide incorporation event; assigning the groups torespective nucleotides to produce nucleotide assignments for the groups;and assigning the points to nucleotides based on the one or morecriteria and the nucleotide assignments for the groups.
 52. Thenon-transitory computer readable storage medium of claim 46, wherein thetemporal characteristic and the intensity characteristic are used toperform identifying individual nucleotides, wherein the identifying ofthe individual nucleotides comprises assigning the nucleotideincorporation events to nucleotides by evaluating the temporalcharacteristic and the intensity characteristic in view of storedcriteria for the sequencing instrument, and distinguishing between thecharacteristics of the light for the luminescent labels.
 53. Thenon-transitory computer readable storage medium of claim 52, wherein thestored criteria comprises one or more boundaries between characteristicsof the luminescent labels for different nucleotides, and the assigningof the nucleotide incorporation events comprises comparing a pointrepresenting the temporal characteristic and the intensitycharacteristic with the one or more boundaries.
 54. The non-transitorycomputer readable storage medium of claim 52, wherein the storedcriteria comprises centroids of groups of points, each groupcorresponding to a respective nucleotide, and the assigning of thenucleotide incorporation events comprises: determining distances betweena point representing the temporal characteristic and the intensitycharacteristic for an incorporation event to the centroids; andassigning the nucleotide incorporation event to a nucleotide with acentroid closest to the point.
 55. The non-transitory computer readablestorage medium of claim 47, wherein the temporal characteristic and theintensity characteristic are used to perform identifying of individualnucleotides, wherein the identifying the individual nucleotidescomprises: determining one or more criteria distinguishing groups ofpoints representing the characteristics of the nucleotide incorporationevents, individual points representing the temporal characteristic andthe intensity characteristic for a corresponding nucleotideincorporation event; assigning the groups to respective nucleotides toproduce nucleotide assignments for the groups; and assigning the pointsto nucleotides based on the one or more criteria and the nucleotideassignments for the groups.